CMP-dependent sialidase activity

ABSTRACT

The properties of certain glycosyltransferase variants having N-terminal truncation deletions or internal deletions are disclosed. Particularly, mutants that exhibit α-2,6-sialyltransferase enzymatic activity in the presence of CMP-activated sialic acid as co-substrate, and in the presence of a suitable acceptor site, are disclosed. A fundamental finding documented in the present disclosure is that enzymes are not only capable of catalyzing transfer of a sialidyl moiety but they are also capable of catalyzing hydrolytic cleavage of terminally bound sialic acid from a glycan.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 15/629,040 (issued as U.S. Pat. No. 11,078,511) filed Jun. 21, 2017,which is a continuation application of International Application No.PCT/EP2015/080741 filed Dec. 21, 2015, claiming priority to EuropeanApplication No. 14199648.8 filed Dec. 22, 2014, the disclosures of whichare hereby incorporated by reference in their entirety.

BACKGROUND

The present disclosure is directed to the properties of certainglycosyltransferase variants having N-terminal truncation deletions orinternal deletions. Any of the mutants disclosed in here exhibitα-2,6-sialyltransferase enzymatic activity in the presence ofCMP-activated sialic acid as co-substrate, and in the presence of asuitable acceptor site. A fundamental finding documented in the presentdisclosure is that such enzymes are not only capable of catalyzingtransfer of a sialidyl moiety but they are also capable of catalyzinghydrolytic cleavage of terminally bound sialic acid from a glycan.Particularly it was found that in the presence ofcytidine-5′-monophosphate (CMP) glycosyltransferase activity isinhibited, and sialidase activity is stimulated. Sialidase activity wasfound to be dependent on the presence of a particular stretch of aminoacids (position 90 to 108) in the polypeptide sequence of the reference(wildtype) hST6Gal-I polypeptide. Deletion of this sequence portion inan N-terminal truncation variant was found to abolish sialidaseactivity, notably both in the presence and in the absence of CMP. Thus,disclosed are compositions, uses and methods employing the CMP-mediatedfeed-back regulation documented herein.

Contrary to previous findings α-2,6-sialyltransferase mutants were foundto exhibit sialidase enzymatic activity, particularly (but not limitedto) a variant of human β-galactoside-α,-2,6-sialyltransferase I(hST6Gal-I; wildtype amino acid sequence see SEQ ID NO:1) with atruncation deletion involving the first 89 N-terminal amino acids of therespective wild-type polypeptide (i.e. a mutant with the amino acidsequence of SEQ ID NO:2). A fundamental finding documented in thepresent disclosure is that this mutant enzyme is not only capable ofcatalyzing transfer of a sialidyl moiety; in fact, theα-2,6-sialyltransferase variant is also capable of catalyzing hydrolyticcleavage of terminally bound sialic acid from a glycan. The presentdisclosure further reports the unexpected observation of feed-backinhibition. Particularly it was found that in the presence ofcytidine-5′-monophosphate (CMP) glycosyltransferase activity isinhibited, and sialidase activity is stimulated. Even more surprising,not only the deletion mutant involving the first 89 N-terminal aminoacids but also other N-terminal truncation variants of humanβ-galactoside-α-2,6-sialyltransferase I (hST6Gal-I) were found toexhibit sialidase enzymatic activity. However, sialidase activity wasfound to be dependent on the presence of a particular stretch of aminoacids (position 90 to 108, see FIG. 1 ) in the polypeptide sequence ofthe reference (wildtype) hST6Gal-I polypeptide according to SEQ IDNO: 1. Deletion of this sequence portion in an N-terminal truncationvariant was found to abolish sialidase activity, notably both in thepresence and in the absence of CMP. Thus, disclosed are compositions,uses and methods employing the CMP-mediated feed-back regulationdocumented herein, particularly directed to controlled hydrolysis of theα2,6 glycosidic bond in aN-acetylneuraminyl-α2,6-β-D-galactosyl-1,4-N-acetyl-β-D-glucosaminemoiety. Further, compositions, uses and methods with a CMP-insensitivehST6Gal-I are disclosed.

Transferases (EC 2) catalyze transfer of a functional group from onesubstance to another. Glycosyltransferases, a superfamily of enzymes,are involved in synthesizing the carbohydrate portions of glycoproteins,glycolipids and glycosaminoglycans. Specific glycosyltransferasessynthesize oligosaccharides by the sequential transfer of themonosaccharide moiety of an activated sugar donor to an acceptormolecule. Hence, a “glycosyltransferase” catalyzes the transfer of asugar moiety from its nucleotide donor to an acceptor moiety of apolypeptide, lipid, glycoprotein or glycolipid. This process is alsoknown as “glycosylation”. A carbohydrate portion which is structuralpart of e.g. a glycoprotein is also referred to as “glycan”. Glycansconstitute the most prevalent of all known post-translational proteinmodifications. Glycans are involved in a wide array of biologicalrecognition processes as diverse as adhesion, immune response, neuralcell migration and axonal extension. As structural part of glycoproteinsglycans also have a role in protein folding and the support of proteinstability and biological activity.

In glycosyltransferase catalysis, the monosaccharide units glucose(Glc), galactose (Gal), N-acetylglucosamine (GlcNAc),N-acetylgalactosamine (GalNAc), glucuronic acid (GlcUA), galacturonicacid (GalUA) and xylose are activated as uridine diphosphate (UDP)-α-Dderivatives; arabinose is activated as a UDP-β-L derivative; mannose(Man) and fucose are activated as GDP-α-D and GDP-β-L derivatives,respectively; and sialic acid (=β-D-Neu5Ac; =Neu5Ac; =SA; =NANA) isactivated as a CMP derivative of sialic acid. CMP-activated sialic acid(=CMP-β-D-Neu5Ac, see below) appears to be the only naturally occurringnucleotide sugar in the form of a nucleotide monophosphate.

Many different glycosyltransferases contribute to the synthesis ofglycans. The structural diversity of carbohydrate portions ofglycoproteins is particularly large and is determined by complexbiosynthetic pathways. In eukaryotes the post-translational biosynthesisof the glycan-part of glycoproteins takes place in the lumen of theendoplasmatic reticulum (“ER”) and the Golgi apparatus. A single(branched or linear) carbohydrate chain of a glycoprotein is typically aN- or an O-linked glycan. During post-translational processing,carbohydrates are typically connected to the polypeptide via asparagine(“N-linked glycosylation”), or via serine or threonine (“O-linkedglycosylation”). Synthesis of a glycan, no matter whether N- or O-linked(=“N-/O-linked”) is effected by the activity of several differentmembrane-anchored glycosyltransferases. A glycoprotein may comprise oneor more glycan-connected amino acids (=“glycosylation sites”). Aspecific glycan structure may be linear or branched. Branching is anotable feature of carbohydrates which is in contrast to the linearnature typical for DNA, RNA, and polypeptides. Combined with the largeheterogeneity of their basic building blocks, the monosaccharides,glycan structures exhibit high diversity. Furthermore, in members of aparticular glycoprotein species the structure of a glycan attached to aparticular glycosylation site may vary, thus resulting inmicroheterogeneity of the respective glycoprotein species, i.e. in aspecies sharing the same amino acid sequence of the polypeptide portion.

A sialyltransferase (=“ST”) is a glycosyltransferase that catalyzestransfer of a sialic acid residue from a donor compound to (i) aterminal monosaccharide acceptor group of a glycolipid or a ganglioside,or (ii) to a terminal monosaccharide acceptor group of an N-/O-linkedglycan of a glycoprotein. For the purpose of the present disclosure, thedonor compound is also referred to as “co-substrate”. For mammaliansialyltransferases including human ST species there is a common donorcompound which is cytidine-5′-monophospho-N-acetylneuraminic acid(=CMP-β-D-Neu5Ac=CMP-Neu5Ac=CMP-NANA; =CMP-sialic acid; =CMP-SA). Wellknown to the skilled person, CMP-sialic acid is a specific embodiment ofa donor compound for a sialyltransferase; further, there existfunctional equivalents including but not limited toCMP-9-fluoresceinyl-sialic acid. Transfer and covalent coupling of asialic acid residue (or the functional equivalent thereof) to a receptorsite is also referred to as “sialylating” and “sialylation”.

In the glycan structure of a sialylated glycoprotein the (one or more)sialyl moiety (moieties) is (are) usually found in the terminal positionof an oligosaccharide. Thus, depending on the amount of sialylatedsites, one or more sialic acid residue(s) can form part of a glycanmoiety of a given glycoprotein. Owing to the terminal, i.e. exposedposition, sialic acid can participate in many different biologicalrecognition phenomena and serve in different kinds of biologicalinteractions. In a glycoprotein more than one sialylation site may bepresent, i.e. a site capable of serving as a substrate for asialyltransferase and being an acceptor group suitable for the transferof a sialic acid residue. Such more than one site can in principle bethe termini of a plurality of linear glycan portions anchored atdifferent glycosylation sites of the glycoprotein. Additionally, abranched glycan may have a plurality of sites where sialylation canoccur.

According to current knowledge, a terminal sialic acid residue can befound (i) α2→3 (α2,3) linked to galactosyl-R, (ii) α2→6 (α2,6) linked togalactosyl-R, (iii) α2→6 (α2,6) linked to N-acetylgalactosaminidyl-R,(iv) α2→6 (α2,6) linked to N-acetylglucosaminidyl-R, and (v) α2-8/9(α2,8/9) linked to sialidyl-R, wherein —R denotes the rest of theacceptor substrate moiety. Hence, a sialyltransferase active in thebiosynthesis of sialylconjugates is generally named and classifiedaccording to its respective monosaccharide acceptor substrate andaccording to the 3, 6 or 8/9 position of the glycosidic bond itcatalyzes. Accordingly, in the literature known to the art, e.g. inPatel R Y, et al, Glycobiology 16 (2006) 108-116, reference toeukaryotic sialyltransferases is made such as (i) ST3Gal, (ii) ST6Gal,(iii) ST6GalNAc, or (v) ST8Sia, depending on the hydroxyl position ofthe acceptor sugar residue to which the Neu5Ac residue is transferredwhile forming a glycosidic bond. Reference to sialyltransferases in amore generic way can also be made e.g. as ST3, ST6, ST8; thus, “ST6”specifically encompasses the sialyltransferases catalyzing an α2,6sialylation.

The disaccharide moiety β-D-galactosyl-1,4-N-acetyl-β-D-glucosamine(=Galβ1,4GlcNAc) is a frequent terminal residue of the antennae ofN-linked glycans of glycoproteins, but may be also present in O-linkedglycans and in glycolipids. In addition, a terminal Galβ1,4GlcNAc moietycan be generated in certain target glycoproteins as a result ofgalactosyltransferase enzymatic activity. The enzymeβ-galactoside-α,2,6-sialyltransferase (=“ST6Gal”) is able to catalyzeα2,6-sialylation of a terminal Galβ1,4GlcNAc acceptor moiety of a glycanor a branch of a glycan, also known to the art as “antenna”. For generalaspects thereof, reference is made to the document of DallOlio F.Glycoconjugate Journal 17 (2000) 669-676. In human and in other mammalsthere appear to be several species (isozymes) of ST6Gal. The presentdisclosure particularly discloses humanβ-galactoside-α-2,6-sialyltransferase I (=hST6Gal-I; EC 2.4.99.1according to IUBMB Enzyme Nomenclature) and variants thereof, but is notlimited thereto.

The ST6 group of sialyltransferases comprises two subgroups, ST6Gal andST6GalNAc. The activity of ST6Gal enzymes catalyzes transfer of a Neu5Acresidue to the C6 hydroxyl group of a free galactosyl residue being partof terminal Galβ1,4GlcNAc in a glycan or an antenna of a glycan, therebyforming in the glycan a terminal sialic acid residue α2→6 linked to thegalactosyl residue of the Galβ1,4GlcNAc moiety. The resulting newlyformed terminal moiety in the glycan is Neu5Acα,2,6Galβ1,4GlcNAc.

The wild-type polypeptide of humanβ-galactoside-α,-2,6-sialyltransferase I (hST6Gal-I) at the time offiling of the present document was disclosed as “UniProtKB/Swiss-Prot:P15907.1” in the publically accessible NCBI database(www.ncbi.nlm.nih.gov/protein/115445). Further information includingcoding sequences are provided as hyperlinks compiled within the databaseentry “Gene ID: 6480” (www.ncbi.nlm.nih.gov/gene/6480).

Mammalian sialyltransferases share with other mammalian Golgi-residentglycosyltransferases a so-called “type II architecture” with (i) a shortcytoplasmic N-terminal tail, (ii) a transmembrane fragment followed by(iii) a stem region of variable length and (iv) a C-terminal catalyticdomain facing the lumen of the Golgi apparatus (Donadio S. et al. inBiochimie 85 (2003) 311-321). Accordingly, a “soluble” sialyltransferasewith an N-terminal truncation deletion lacks at least the elements (i)and (ii) of the type II architecture. Mammalian sialyltransferasesappear to display significant sequence homology in their catalyticdomain. Recent data regarding structure and function of hST6Gal-I aredisclosed in Kuhn B. et al. (Biol. Crystallography D69 (2013)1826-1838).

In certain mammals including mouse, rat and humans, ST6Gal haswidespread tissue distribution. It is particularly abundant in liver,the major site of serum glycoprotein synthesis (Weinstein J. et al. J.Biol. Chem 257 (1982) 13835-13844). On the one hand sialyltransferaseexists predominantly in a membrane-bound form within the Golgi andtrans-Golgi network where it participates in the posttranslationalmodification of newly synthesized secretory or cell surfaceglycoproteins. On the other hand, a soluble form of ST6Gal-I exists inthe serum (Kim Y. S. et al Biochim. Biophys Acta 244 (1971) 505-512;Dalziel M. et al. Glycobiology 9 (1999) 1003-1008) and predominantly isderived from the liver (Kaplan, H. A. et al. J. Biol. Chem. 258 (1983)11505-11509; van Dijk, W. et al. Biochem. Cell. Biol. 64 (1986) 79-84;Dalziel M. et al. (supra)) by a proteolytic event that liberates thecatalytic domain from its membrane anchor (Kaplan et al., supra;Weinstein, J. et al. J. Biol. Chem. 262 (1987) 17735-17743). Thus, anyvariant of an originally membrane-anchored glycosyltransferase with anN-terminal truncation comprising the membrane anchor is encompassed bythe term “soluble variant”. Soluble variants can exemplarily begenerated by proteolytically removing the portion with the membraneanchor from the protein, or by expressing a variant nucleic acidsequence encoding a N-terminally truncated form of the original proteinwherein the truncation includes the membrane anchor (transmembranefragment, element (ii) of the type II architecture).

Donadio S. et al. (supra) recombinantly expressed several N-terminallytruncated variants of hST6Gal-I without the membrane anchor in CHOcells. The authors found that N-terminal deletions comprising the first35, 48, 60 and 89 amino acids yielded variants of hST6Gal-I which wereenzymatically active and capable of transferring sialic acid toexogenous acceptors.

Glycosylation is an important posttranslational modification of proteinsinfluencing protein folding, stability and regulation of the biologicalactivity. The sialyl residue is usually exposed at the terminal positionof an N-glycan and therefore, a major contributor to biologicalrecognition and ligand function. As an important example, IgG withglycans featuring terminal sialic acid residues were found to inducereduced inflammatory response and showed an increase in serum half-life.Therefore, use of glycosyltransferases for enzymatic synthesis ofdefined glycan structures is becoming an engineering tool towards directin vitro N-glycosylation of therapeutic proteins, and particularlytherapeutic monoclonal antibodies.

Since glycosyltransferases of prokaryotic origin usually do not act oncomplex glycoprotein structures, sialyltransferases of mammalian originare preferred for in vitro glycoengineering purposes. For example, Barbet al. (2009) prepared highly sialylated forms of the Fc fragment ofimmunoglobulin G using isolated human ST6Gal-I. However, the access torecombinant hST6Gal-I for such applications is still limited due to lowexpression yield and/or poor activity of hST6Gal-I recombinantlyexpressed in various hosts (methylotrophic yeast Pichia pastoris,cultured Spodoptera frugiperda cells, E. coli-based expression systems).

Kleineidam R. G. et al. Glycoconjugate Journal (1997) 14: 57-66 disclosea number of inhibitors of α-2,6-sialyltransferase from rat liver.Specifically, 70%, 40%, 39% and 71% inhibition ofα-2,6-sialyltransferase was observed in the presence of Cytidine,2′-CMP, 3′-CMP and 5′-CMP, respectively, wherein each inhibitor wastested at a concentration of 0.25 mM.

While the use of mammalian glycosyltransferases for in vitro sialylatinga glycosylated target molecule such as a glycoprotein or a glycolipid isknown to the art, the opposite reaction (sialidase activity, hydrolyticcleavage of a terminal sialyl residue from a glycan moiety) is typicallyprovided by a neuraminidase, so far. The original finding by the presentinventors is, however, that a soluble variant of a sialyltransferase ofmammalian origin displays sialidase activity in the presence of CMP. Infact, the specific example of a soluble variant of humanp-galactoside-α-2,6-sialyltransferase I lacking the transmembrane domainby means of a N-terminal truncation can be used for both, (i)sialylation of a target glycoprotein and (ii) hydrolytic cleavage ofsialyl residues from a sialylated target glycoprotein. Depending on thepresence of CMP and the interaction of CMP with the soluble variant,sialylation can be controlled quantitatively. In specific embodimentsinvolving target molecules with two or more antennal glycan acceptorsites, the present disclosure provides means, methods and conditionsallowing to sialylate just one out of the several acceptor sites, aswell as sialylating two or more, or even all acceptor sites of thetarget molecule.

This paves the way for a number of different approaches, particularly inthe field of in vitro glycoengineering of immunoglobulins, and also ofother glycosylated target molecules.

SUMMARY

In a first aspect and a specific embodiment of all other aspects asdisclosed herein there is disclosed an aqueous composition comprising

-   (a) a soluble human β-galactoside-α-2,6-sialyltransferase I    comprising the amino acid motif from position 90 to position 108 in    SEQ ID NO:1;-   (b) cytidine-5′-monophospho-N-acetylneuraminic acid, or a functional    equivalent thereof,-   (c) a glycosylated target molecule selected from a glycoprotein and    a glycolipid, the target molecule comprising one or more antenna(e),    at least one antenna having as a terminal structure a    β-D-galactosyl-1,4-N-acetyl-β-D-glucosamine moiety with a hydroxyl    group at the C6 position in the galactosyl residue;-   (d) an aqueous solution permitting glycosyltransferase enzymatic    activity;    wherein the aqueous composition further comprises an enzyme capable    of hydrolyzing the phosphoester bond in 5′-cytidine-monophosphate    under conditions permitting glycosyltransferase enzymatic activity.

In a second aspect and a specific embodiment of all other aspects asdisclosed herein there is disclosed the use of an enzyme capable of thehydrolyzing phosphoester bond in 5′-cytidine-monophosphate formaintaining sialyltransferase enzymatic activity and/or inhibitingsialidase enzymatic activity in a composition according to the firstaspect.

In a third aspect and a specific embodiment of all other aspects asdisclosed herein there is disclosed a method of producing in vitro asialylated target molecule, the method comprising the steps of

-   (a) providing an aqueous composition according to any of the claims    1 and 2;-   (b) forming one or more terminal antennal    N-acetylneuraminyl-α2,6-β-D-galactosyl-1,4-N-acetyl-β-D-glucosamine    residue(s) by incubating the aqueous composition of step (a),    thereby reacting cytidine-5′-monophospho-N-acetylneuraminic acid, or    a functional equivalent thereof, as co-substrate, thereby forming    5′-cytidine-monophosphate;-   (c) hydrolyzing the phosphoester bond of the    5′-cytidine-monophosphate formed in step (b), thereby reducing    5′-cytidine-monophosphate-mediated inhibition, thereby maintaining    the activity of the soluble human    β-galactoside-α,-2,6-sialyltransferase I;    thereby producing in vitro the sialylated target molecule.

In a fourth aspect and a specific embodiment of all other aspects asdisclosed herein there is disclosed a preparation of sialylatedimmunoglobulins, each immunoglobulin having a plurality of acceptorsites for human β-galactoside-α-2,6-sialyltransferase I, wherein lessthan about 25% of the acceptor sites in the preparation of sialylatedimmunoglobulins are not sialylated, and about 75% or more aresialylated, wherein the preparation is obtained by a method according tothe third aspect.

In a fifth aspect and a specific embodiment of all other aspects asdisclosed herein there is disclosed the use of a soluble humanβ-galactoside-α,-2,6-sialyltransferase I comprising the amino acid motiffrom position 90 to position 108 in SEQ ID NO:1 for in vitro hydrolyzingin the presence of 5′-cytidine-monophosphate the α2,6 glycosidic bond ina N-acetylneuraminyl-α,2,6-β-D-galactosyl-1,4-N-acetyl-β-D-glucosaminemoiety, the moiety being a terminal structure of a glycan in asialylated glycoprotein or glycolipid.

DESCRIPTION OF THE FIGURES

FIG. 1 Representation of the amino acid sequence of the wild-typehST6Gal-I polypeptide (SEQ ID NO:1), and the N-terminal portions thereofwhich are truncated in the deletion variants as disclosed herein. Thedeleted positions in the truncations are symbolized by “X”. Underlinedare the amino acid at positions 90-108 (SEQ ID NO:1) found to beessential for CMP-induced Sialidase activity.

FIG. 2 SDS-PAGE gel after electrophoresis and staining of the Δ89hST6Gal-I variant transiently expressed in and secreted from HEK cells.Lane 1 shows a size-standard, molecular weights in kDa according to thestandard are indicated to the left. Lane 2: Purified Δ89 hST6Gal-Itruncation variant (5 μg of protein were loaded on the gel).

FIG. 3 SDS gel after electrophoresis and staining of the Δ108 hST6Gal-Ivariant transiently expressed in and secreted from HEK cells. Lane 1shows a size-standard, molecular weights in kDa according to thestandard are indicated to the left. Lane 2: Δ108 hST6Gal-I truncationvariant (5 μg of protein were loaded on the gel).

FIG. 4 Time course of sialylation of IgG4 MAB using recombinant Δ89hST6Gal-I.

FIG. 5 Kinetics of formation of G2+2SA and G2+1SA, catalyzed byrecombinant Δ89 hST6Gal-I, as shown by mass spectra taken as a basis fordetermination of the relative content of the different sialylated targetmolecule species.

FIG. 6 Inhibition of sialidase activity of recombinant Δ89 hST6Gal-I byCTP. The relative content of antibodies with glycan with terminalgalactose residues (G2+0SA, “asialo”), mono-sialylated glycan (G2+1SA)and bi-sialylated glycan (G2+2SA) is shown for different time points.

FIG. 7 CMP-dependent sialidase activity of Δ89 hST6Gal-I: Incubation ofpurified IgG1 MAB G2+2SA with Δ89 hST6Gal-I in the absence of CMP. Therelative content of antibodies with glycans with terminal galactoseresidues (G2+0SA), monosialylated glycan (G2+1SA) and disialylatedglycan (G2+2SA) are shown for different time points.

FIG. 8 CMP-dependent sialidase activity of Δ89 hST6Gal-I: Incubation ofpurified IgG1 MAB G2+2SA with Δ89 hST6Gal-I in the presence of CMP. Therelative content of antibodies with glycans with terminal galactoseresidues (G2+0SA), monosialylated glycan (G2+1SA) and disialylatedglycan (G2+2SA) are shown for different time points.

FIG. 9 CMP-dependent sialidase activity of Δ89 hST6Gal-I: Incubation ofpurified IgG1 MAB G2+2SA with Δ108 hST6Gal-I in the presence of CMP. Therelative content of antibodies with glycans with terminal galactoseresidues (G2+0SA), monosialylated glycan (G2+1SA) and disialylatedglycan (G2+2SA) are shown for different time points.

FIG. 10 CMP-dependent sialidase activity of Δ89 hST6Gal-I: Incubation ofpurified IgG1 MAB G2+2SA with delta57ST3-Gal-I in the presence of CMP.The relative content of antibodies with glycans with terminal galactoseresidues (G2+0SA), monosialylated glycan (G2+1SA) and disialylatedglycan (G2+2SA) are shown for different time points.

FIG. 11 Sialylation of IgG4 MAB with Δ89 hST6Gal-I in the absence orpresence of 5′-nucleotidase CD73 in the sialylation reaction mixture.The relative content of antibodies with glycans with just terminalgalactose residues (G2+0SA), monosialylated glycan (G2+1SA) anddisialylated glycan (G2+2SA) is shown. The amount of 5′-nucleotidaseused was 0-0.5 μg. Negative control: 0 μg 5′-nucleotidase CD73.

FIG. 12 Sialylation of IgG4 MAB with Δ89 hST6Gal-I in the absence orpresence of 5′-nucleotidase CD73 in the sialylation reaction mixture.The relative content of antibodies with glycans with just terminalgalactose residues (G2+0SA), monosialylated glycan (G2+1SA) anddisialylated glycan (G2+2SA) is shown. Sialylation reaction mixture with0.1 μg 5′-nucleotidase CD73.

FIG. 13 Sialylation of IgG4 MAB with Δ89 hST6Gal-I in the absence orpresence of 5′-nucleotidase CD73 in the sialylation reaction mixture.The relative content of antibodies with glycans with just terminalgalactose residues (G2+0SA), monosialylated glycan (G2+1SA) anddisialylated glycan (G2+2SA) is shown. Sialylation reaction mixture with0.25 μg 5′-nucleotidase CD73.

FIG. 14 Sialylation of IgG4 MAB with Δ89 hST6Gal-I in the absence orpresence of 5′-nucleotidase CD73 in the sialylation reaction mixture.The relative content of antibodies with glycans with just terminalgalactose residues (G2+0SA), monosialylated glycan (G2+1SA) anddisialylated glycan (G2+2SA) is shown. Sialylation reaction mixture with0.5 μg 5′-nucleotidase CD73.

FIG. 15 Sialylation of IgG4 MAB with Δ89 hST6Gal-I in the absence orpresence of alkaline phosphatase in the sialylation reaction mixture.The relative content of antibodies with glycans with just terminalgalactose residues (G2+0SA), monosialylated glycan (G2+1SA) anddisialylated glycan (G2+2SA) is shown. The amount of 5′-nucleotidaseused was 0-100 μg. Negative control: 0 μg alkaline phosphatase.

FIG. 16 Sialylation of IgG4 MAB with Δ89 hST6Gal-I in the absence orpresence of alkaline phosphatase in the sialylation reaction mixture.The relative content of antibodies with glycans with just terminalgalactose residues (G2+0SA), monosialylated glycan (G2+1SA) anddisialylated glycan (G2+2SA) is shown. Sialylation reaction mixture with1 μg alkaline phosphatase.

FIG. 17 Sialylation of IgG4 MAB with Δ89 hST6Gal-I in the absence orpresence of alkaline phosphatase in the sialylation reaction mixture.The relative content of antibodies with glycans with just terminalgalactose residues (G2+0SA), monosialylated glycan (G2+1SA) anddisialylated glycan (G2+2SA) is shown. Sialylation reaction mixture with5 μg alkaline phosphatase.

FIG. 18 Sialylation of IgG4 MAB with Δ89 hST6Gal-I in the absence orpresence of alkaline phosphatase in the sialylation reaction mixture.The relative content of antibodies with glycans with just terminalgalactose residues (G2+0SA), monosialylated glycan (G2+1SA) anddisialylated glycan (G2+2SA) is shown. Sialylation reaction mixture with10 μg alkaline phosphatase.

FIG. 19 Sialylation of IgG4 MAB with Δ89 hST6Gal-I in the absence orpresence of alkaline phosphatase in the sialylation reaction mixture.The relative content of antibodies with glycans with just terminalgalactose residues (G2+0SA), monosialylated glycan (G2+1SA) anddisialylated glycan (G2+2SA) is shown. Sialylation reaction mixture with100 μg alkaline phosphatase.

DETAILED DESCRIPTION

The terms “a”, “an” and “the” generally include plural referents, i.e.“one or more”, unless the context clearly indicates otherwise. As usedherein, “plurality” is understood to mean more than one. For example, aplurality refers to at least two, three, four, five, or more. Unlessspecifically stated or obvious from context, as used herein, the term“or” is understood to be inclusive.

Unless specifically stated or obvious from context, as used herein, theterm “about” is understood as within a range of normal tolerance in theart, for example within 2 standard deviations of the mean. About can beunderstood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%,0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear fromcontext, all numerical values provided herein can be modified by theterm “about”.

The term “amino acid” generally refers to any monomer unit that can beincorporated into a peptide, polypeptide, or protein. As used herein,the term “amino acid” includes the following twenty natural orgenetically encoded alpha-amino acids: alanine (Ala or A), arginine (Argor R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine (Cys orC), glutamine (Gln or Q), glutamic acid (Glu or E), glycine (Gly or G),histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine(Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline(Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp orW), tyrosine (Tyr or Y), and valine (Val or V). In cases where “X”residues are undefined, these should be defined as “any amino acid.” Thestructures of these twenty natural amino acids are shown in, e.g.,Stryer et al., Biochemistry, 5th ed., Freeman and Company (2002).Additional amino acids, such as selenocysteine and pyrrolysine, can alsobe genetically coded for (Stadtman (1996) “Selenocysteine,” Annu RevBiochem. 65:83-100 and Ibba et al. (2002) “Genetic code: introducingpyrrolysine,” Curr Biol. 12(13):R464-R466). The term “amino acid” alsoincludes unnatural amino acids, modified amino acids (e.g., havingmodified side chains and/or backbones), and amino acid analogs. See,e.g., Zhang et al. (2004) “Selective incorporation of5-hydroxytryptophan into proteins in mammalian cells,” Proc. Natl. Acad.Sci. U.S.A. 101(24):8882-8887, Anderson et al. (2004) “An expandedgenetic code with a functional quadruplet codon” Proc. Natl. Acad. Sci.U.S.A. 101(20):7566-7571, Ikeda et al. (2003) “Synthesis of a novelhistidine analogue and its efficient incorporation into a protein invivo,” Protein Eng. Des. Sel. 16(9):699-706, Chin et al. (2003) “AnExpanded Eukaryotic Genetic Code,” Science 301(5635):964-967, James etal. (2001) “Kinetic characterization of ribonuclease S mutantscontaining photoisomerizable phenylazophenylalanine residues,” ProteinEng. Des. Sel. 14(12):983-991, Kohrer et al. (2001) “Import of amber andochre suppressor tRNAs into mammalian cells: A general approach tosite-specific insertion of amino acid analogues into proteins,” Proc.Natl. Acad. Sci. U.S.A. 98(25):14310-14315, Bacher et al. (2001)“Selection and Characterization of Escherichia coli Variants Capable ofGrowth on an Otherwise Toxic Tryptophan Analogue,” J. Bacteriol.183(18):5414-5425, Hamano-Takaku et al. (2000) “A Mutant Escherichiacoli Tyrosyl-tRNA Synthetase Utilizes the Unnatural Amino AcidAzatyrosine More Efficiently than Tyrosine,” J. Biol. Chem.275(51):40324-40328, and Budisa et al. (2001) “Proteins with{beta}-(thienopyrrolyl)alanines as alternative chromophores andpharmaceutically active amino acids,” Protein Sci. 10(7):1281-1292. Tofurther illustrate, an amino acid is typically an organic acid thatincludes a substituted or unsubstituted amino group, a substituted orunsubstituted carboxy group, and one or more side chains or groups, oranalogs of any of these groups. Exemplary side chains include, e.g.,thiol, seleno, sulfonyl, alkyl, aryl, acyl, keto, azido, hydroxyl,hydrazine, cyano, halo, hydrazide, alkenyl, alkynl, ether, borate,boronate, phospho, phosphono, phosphine, heterocyclic, enone, imine,aldehyde, ester, thioacid, hydroxylamine, or any combination of thesegroups. Other representative amino acids include, but are not limitedto, amino acids comprising photoactivatable cross-linkers, metal bindingamino acids, spin-labeled amino acids, fluorescent amino acids,metal-containing amino acids, amino acids with novel functional groups,amino acids that covalently or noncovalently interact with othermolecules, photocaged and/or photoisomerizable amino acids, radioactiveamino acids, amino acids comprising biotin or a biotin analog,glycosylated amino acids, other carbohydrate modified amino acids, aminoacids comprising polyethylene glycol or polyether, heavy atomsubstituted amino acids, chemically cleavable and/or photocleavableamino acids, carbon-linked sugar-containing amino acids, redox-activeamino acids, amino thioacid containing amino acids, and amino acidscomprising one or more toxic moieties.

The term “protein” refers to a polypeptide chain (amino acid sequence)as a product of the ribosomal translation process, wherein thepolypeptide chain has undergone posttranslational folding processesresulting in three-dimensional protein structure. The term “protein”also encompasses polypeptides with one or more posttranslationalmodifications such as (but not limited to) glycosylation,phosphorylation, acetylation and ubiquitination.

Any protein as disclosed herein, particularly recombinantly producedprotein as disclosed herein, may in a specific embodiment comprise a“protein tag” which is a peptide sequence genetically grafted onto therecombinant protein. A protein tag may comprise a linker sequence with aspecific protease cleavage site to facilitate removal of the tag byproteolysis. As a specific embodiment, an “affinity tag” is appended toa target protein so that the target can be purified from its crudebiological source using an affinity technique. For example, the sourcecan be a transformed host organism expressing the target protein or aculture supernatant into which the target protein was secreted by thetransformed host organism. Specific embodiments of an affinity taginclude chitin binding protein (CBP), maltose binding protein (MBP), andglutathione-S-transferase (GST). The poly(His) tag is a widely-usedprotein tag which facilitates binding to certain metal chelatingmatrices.

Each of the terms “chimeric protein”, “fusion protein” or “fusionpolypeptide” equally refers to a protein whose amino acid sequencerepresents a fusion product of subsequences of the amino acid sequencesfrom at least two distinct proteins. A fusion protein typically is notproduced by direct manipulation of amino acid sequences, but, rather, isexpressed from a “chimeric” gene that encodes the chimeric amino acidsequence.

The term “recombinant” refers to an amino acid sequence or a nucleotidesequence that has been intentionally modified by recombinant methods. Bythe term “recombinant nucleic acid” herein is meant a nucleic acid,originally formed in vitro, in general, by the manipulation of a nucleicacid by endonucleases, in a form not normally found in nature. Thus anisolated, mutant DNA polymerase nucleic acid, in a linear form, or anexpression vector formed in vitro by ligating DNA molecules that are notnormally joined, are both considered recombinant for the purposes ofthis invention. It is understood that once a recombinant nucleic acid ismade and reintroduced into a host cell, it will replicatenon-recombinantly, i.e., using the in vivo cellular machinery of thehost cell rather than in vitro manipulations; however, such nucleicacids, once produced recombinantly, although subsequently replicatednon-recombinantly, are still considered recombinant for the purposes ofthe invention. A “recombinant protein” or “recombinantly producedprotein” is a protein made using recombinant techniques, i.e., throughthe expression of a recombinant nucleic acid as depicted above.

The term “host cell” refers to both single-cellular prokaryote andeukaryote organisms (e.g., mammalian cells, insect cells, bacteria,yeast, and actinomycetes) and single cells from higher order plants oranimals when being grown in cell culture.

The term “glycosylation” denotes the chemical reaction of covalentlycoupling a glycosyl residue to an acceptor group. One specific acceptorgroup is a hydroxyl group, e.g. a hydroxyl group of another sugar.“Sialylation” is a specific form of glycosylation wherein the acceptorgroup is reacted with a sialic acid (=N-acetylneuraminic acid) residue.Such a reaction is typically catalyzed by a sialyltransferase enzymeusing cytidine-5′-monophospho-N-acetylneuraminic acid as donor compoundor co-substrate.

“Sialylation” is a specific embodiment of a result ofglycosyltransferase enzymatic activity (sialyltransferase enzymaticactivity in the particular case), under conditions permitting the same.

Generally, the skilled person appreciates that the aqueous compositionin which glycosyltransferase enzymatic activity can take place (=underconditions “permitting glycosyltransferase enzymatic activity”) needs tobe buffered using a buffer salt such as Tris, MES, phosphate, acetate,or another buffer salt specifically capable of buffering in the pH rangeof pH 6 to pH 8, more specifically in the range of pH 6 to pH 7, evenmore specifically capable of buffering a solution of about pH 6.5. Thebuffer may further contain a neutral salt such as but not limited toNaCl. Further, in particular embodiments the skilled person may consideradding to the aqueous buffer a salt comprising a divalent cation such asMg²⁺ or Mn²⁺, e.g., but not limited to, MgCl₂ and MnCl₂. In additionalspecific embodiments, the aqueous composition permittingglycosyltransferase enzymatic activity may comprise an antioxidantand/or a surfactant. Conditions permitting glycosyltransferase enzymaticactivity known to the art include ambient (room) temperature, but moregenerally temperatures in the range of 0° C. to 40° C., particularly 10°C. to 30° C., particularly about 20° C. While the above describedconditions provide general conditions permitting such enzymaticactivity, glycosyltransferase activity further requires the presence ofan activated sugar donor (such as specifically CMP-Neu5Ac) as aco-substrate, in addition. However, the term “permittingglycosyltransferase enzymatic activity” is understood as not necessarilyincluding the presence of the co-substrate. Thus, the term “permittingglycosyltransferase enzymatic activity” herein also includes conditionspermitting the hydrolysis (sialidase) activity of a mammalianglycosyltransferase subject of the present disclosure, particularlyhydrolysis activity in the presence of 5′-cytidine-monophosphate (CMP).

The term “glycan” refers to a poly- or oligosaccharide, i.e. to amultimeric compound which upon acid hydrolysis yields a plurality ofmonosachharides. A glycoprotein comprises one or more glycan moietieswhich are covalently coupled to side groups of the polypeptide chain,typically via asparagine or arginine (“N-linked glycosylation”) or viaserine or threonine (“O-linked glycosylation”).

The use of glycosyltransferases for enzymatic synthesis of complexglycan structures is an attractive approach to obtain complex bioactiveglycoproteins. E.g. Barb et al. Biochemistry 48 (2009) 9705-9707prepared highly potent sialylated forms of the Fc fragment ofimmunoglobulin G using isolated human ST6Gal-I. However, growinginterest in the therapeutic application of glycoproteins leads to anincreasing demand of glycosyltransferases including sialyltransferases.Different strategies to increase or modify the sialylation ofglycoproteins were described by Bork K. et al. J. Pharm. Sci. 98 (2009)3499-3508. An attractive strategy is sialylation in vitro ofrecombinantly produced proteins (such as but not limited toimmunoglobulins and growth factors), particularly therapeutic proteins.To this end, several research groups described expression ofsialyltransferases in transformed organisms and purification of therecombinantly produced sialyltransferases. As glycosyltransferases ofprokaryotic origin usually do not act on complex glycoproteins (e.g.antibodies), sialyltransferases from mammalian origin were studied withpreference.

Particular glycoproteins subject to the disclosures and all aspects ofthe present document and the aspects and embodiments herein comprisewithout limitation cell surface glycoproteins and glycoproteins presentin soluble form in serum (“serum glycoprotein”), the glycoproteinsparticularly being of mammalian origin. A “cell surface glycoprotein” isunderstood to be glycoprotein of which a portion is located on and boundto the surface of a membrane, by way of a membrane anchor portion of thesurface glycoprotein's polypeptide chain, wherein the membrane is partof a biological cell. The term cell surface glycoprotein alsoencompasses isolated forms of the cell surface glycoprotein as well assoluble fragments thereof which are separated from the membrane anchorportion, e.g. by proteolytic cleavage or by recombinant production ofsuch soluble fragments. A “serum glycoprotein” is understood as aglycoprotein being present in serum, i.e. a blood protein present in thenon-cellular portion of whole blood, e.g. in the supernatant followingsedimentation of cellular blood components. Without limitation, aspecifically regarded and embodied serum glycoprotein is animmunoglobulin. Particular immunoglobulins mentioned in here belong tothe IgG group (characterized by Gamma heavy chains), specifically any offour the IgG subgroups. For the disclosures, aspects and embodimentsherein the term “serum glycoprotein also encompasses a monoclonalantibody; monoclonal antibodies artificially are well known to the artand can be produced e.g. by hybridoma cells or recombinantly usingtransformed host cells. A further serum specific glycoprotein is acarrier protein such as serum albumin, a fetuin, or another glycoproteinmember of the superfamily of histidine-rich glycoproteins of which thefetuins are members. Further, without limitation, a specificallyregarded and embodied serum glycoprotein regarding all disclosures,aspects and embodiments herein is a glycosylated protein signallingmolecule. A particular molecule of this group is erythropoietin (EPO).

For in vitro engineering of glycoproteins glycosyltransferases can beused as an efficient tool (Weijers 2008). Glycosyltransferases ofmammalian origin are compatible with glycoproteins as substrates whereasbacterial glycosyltransferases usually modify simpler substrates likeoligosaccharides. For this reason synthetic changes in the glycanmoieties of glycoproteins are advantageously made using mammalianglycosyltransferases as tools of choice. However, for a large scaleapplication of glycosyltransferases in glycoengineering availability ofsuitable enzymes in large (i.e. industrial) quantities is required. Thedisclosure herein particularly provides proteins with (i) hST6Gal-Isialyltransferase activity and (ii) sialidase activity which can be usedfor quantitatively controlled in vitro sialylation of targetglycoproteins with one or more accessible galactosyl substratemoiety/moieties.

Importantly, the amino acid motif in humanβ-galactoside-α,-2,6-sialyltransferase I from position 90 to position108 in SEQ ID NO:1 is required for the enzyme to be capable ofexhibiting sialidase activity. At the same time, this amino acid motifis required for the enzyme to interact with 5′-CMP. Very remarkably atruncation deletion mutant, a soluble humanβ-galactoside-α-2,6-sialyltransferase I variant lacking the contiguousN-terminal stretch of the amino acids from position 1 to position 108does not exhibit sialidase activity, not even in the presence of CMP.Thus, it was concluded that the amino acid motif in humanβ-galactoside-α-2,6-sialyltransferase I from position 90 to position 108in SEQ ID NO:1 is essential for these properties to be present.

Suitable targets to treat with sialidase activity include on the onehand asialoglycoproteins, i.e. glycoproteins from which sialic acidresidues have been removed by the action of sialidases. On the otherhand, bi-sialylated glycoproteins may serve as substrate for sialidaseactivity. Very advantageously, asialo-, mono-sialylated andbi-sialylated immunoglobulins are specific substrates, particularlyimmunoglobulins of the IgG class.

While expressing wild-type hST6Gal-I in the methylotrophic yeast Pichiapastoris and having targeted the expressed polypeptide to the secretorypathway of the host organism, different truncated variants ofrecombinantly produced hST6Gal-I were observed. Generally, hST6Gal-Iderived proteins were chromatographically purified and analyzed,particularly by means of mass spectrometry and by way of determining theamino acid sequence from the N-terminus (Edman degradation). By thesemeans truncations, particularly N-terminal truncations of hST6Gal-I werecharacterized in detail.

Several remarkable truncation variants were identified in thesupernatants of transformed Pichia strains. The variants could possiblyresult from site-specific proteolytic cleavage during the course ofsecretion from the yeast cells, or result from endoproteolytic cleavageby one or more extracellular protease(s) present in the supernatant ofcultured Pichia strains.

Each identified truncation variant was given a “delta” (=“Δ”)designation indicating the number of the last amino acid position of therespective truncation deletion, counted from the N-Terminus of thewild-type hST6Gal-I polypeptide according to SEQ ID NO:1 The particularN-terminal truncation variants Δ89 and Δ108 of hST6Gal-I wererecombinantly expressed and studied in more detail.

Expression vectors were constructed for expression of hST6Gal-Iwild-type protein as well as of the Δ89 and Δ108 truncation variants invarious host organisms including prokaryotes such as E. coli andBacillus sp., yeasts such as Saccharomyces cerevisiae and Pichiapastoris, and mammalian cells such as CHO cells and HEK cells. Vectorswith expression constructs for the Δ89 and Δ108 truncation variants ofhST6Gal-I were assembled molecularly thereby providing the means ofrecombinantly producing the Δ89 variant of human ST6Gal-I in severaltransformed host organisms. To facilitate purification of recombinantlyexpressed enzymes, the encoded truncation polypeptides encoded by theconstructs optionally included a N-terminal His-tag, in specificembodiments.

An aspect and a specific embodiment of all other aspects as disclosedherein is a variant mammalian glycosyltransferase capable of catalyzinghydrolysis of the α2,6 glycosidic bond of aN-acetylneuraminyl-α2,6-β-D-galactosyl-1,4-N-acetyl-β-D-glucosaminemoiety of a glycan in a glycoprotein. Particularly, the variantmammalian glycosyltransferase is capable of catalyzing formation of theα2,6 glycosidic bond of aN-acetylneuraminyl-α,2,6-β-D-galactosyl-1,4-N-acetyl-β-D-glucosaminemoiety in a glycoprotein glycan, thereby generating freeN-acetylneuraminic acid. In a specific embodiment of all aspects asdisclosed herein, the variant mammalian glycosyltransferase is a solubleenzyme comprising the amino acid motif from position 90 to position 108in SEQ ID NO:1 disclosing human β-galactoside-α-2,6-sialyltransferase I.Encompassed by the teachings as disclosed in here are homologoussialyltransferases comprising an amino acid motif corresponding to themotif from position 90 to position 108 in SEQ ID NO:1.

In a specific embodiment of all aspects as disclosed herein, the variantmammalian glycosyltransferase capable of catalyzing hydrolysis of theα2,6 glycosidic bond is a mammalian glycosyltransferase is derived, byway of amino acid deletion, from humanβ-galactoside-α,-2,6-sialyltransferase I according to SEQ ID NO:1, saidsequence being truncated by a deletion from the N-terminus. In a furtherspecific embodiment of all aspects as disclosed herein, the truncationdeletion from the N-terminus is the contiguous sequence of position 1 toposition 89 of SEQ ID NO:1.

Another aspect and a specific embodiment of all other aspects asdisclosed herein is a fusion polypeptide comprising a polypeptide of avariant mammalian glycosyltransferase according to any embodiment asdisclosed herein. A fusion protein or fusion polypeptide is a chimericpolypeptide comprising amino acid sequences of two or more polypeptides.The two or more polypeptides may have complementary functions, one ofthe polypeptides may provide a supplementary functional property, or oneof the polypeptides may have a function unrelated to the others in thefusion polypeptide. One or more polypeptides comprising organelletargeting or retention sequences may be fused with a desired polypeptideto target the desired polypeptide to a specific cellular organelle, orretain the desired polypeptide within the cell. One or more polypeptidescomprising a carrier sequence that aids in expression, purificationand/or detection of the fusion polypeptide may be fused with a desiredpolypeptide (e.g., FLAG, a myc tag, a 6×His tag, GST fusions and thelike). Particular fusion partners include N-terminal leader peptidescapable of directing the variant mammalian glycosyltransferase portionof the fusion polypeptide to the secretory pathway of the host organismin which the fusion polypeptide is expressed. Thereby secretion in theextracellular space and the surrounding medium is facilitated. Yet,another aspect and a specific embodiment of all other aspects asdisclosed herein is a nucleotide sequence encoding a variant mammalianglycosyltransferase according to any embodiment as disclosed herein or afusion polypeptide comprising as a portion a variant mammalianglycosyltransferase according to any embodiment as disclosed herein.Importantly, the nucleotide sequence includes the sequence from position90 to position 108 in SEQ ID NO:1, or the homologous equivalent thereofin the case of a sialyltransferase homologous to humanβ-galactoside-α-2,6-sialyltransferase I.

Yet, another aspect and a specific embodiment of all other aspects asdisclosed herein is an expression vector comprising a target gene andsequences facilitating expression of the target gene in a host organismtransformed with the expression vector, wherein the target genecomprises a nucleotide sequence as disclosed herein.

Yet, another aspect and a specific embodiment of all other aspects asdisclosed herein is a transformed host organism, wherein the hostorganism is transformed with an expression vector as disclosed herein.With particular advantage, Human Embryonic Kidney 293 (HEK) cells can beused to practice the teachings as disclosed in here. A particularadvantage of these cells is that they are very suited targets fortransfection followed by subsequent culture and transient expression ofthe target gene. Thus, HEK cells can be efficiently used to producetarget proteins by way of recombinant expression. With great advantage,expression constructs are designed to direct the translation products tothe secretory pathway leading to secretion of the variant mammalianglycosyltransferase or a fusion polypeptide as disclosed herein.Nevertheless, Jurkat, NIH3T3, HeLa, COS and Chinese Hamster Ovary (CHO)cells are well-known alternatives and are included herein as alternativehost organisms for transformation and specific embodiments of allaspects as disclosed herein.

Yet, another aspect and a specific embodiment of all other aspects asdisclosed herein is a method to produce recombinantly a variantmammalian glycosyltransferase, the method comprising the step ofexpressing in a host organism transformed with an expression vector anucleotide sequence encoding a variant mammalian glycosyltransferase asdisclosed herein, wherein a polypeptide is formed, thereby producingvariant mammalian glycosyltransferase.

According to earlier knowledge, N-terminally truncated variants ofglycosyltransferases are advantageously used in vitro due to their lackof transmembrane domains. Thus, such variants are useful for catalyzingand performing glycosyltransferase reactions in solution. It wassurprisingly found and is disclosed herein that particularly theN-terminally truncated variant Δ89 hST6Gal-I displays differentactivities in vitro, e.g. when incubated with glycosylated antibodiesand in the presence of 5′-CMP. Thus, a specific embodiment of thepresent disclosure and all aspects and embodiments herein is a variantmammalian glycosyltransferase capable of catalyzing hydrolysis of a α2,6glycosidic bond of aN-acetylneuraminyl-α2,6-β-D-galactosyl-1,4-N-acetyl-β-D-glucosaminemoiety of a bi-sialylated glycoprotein, i.e. a glycoprotein comprisingtwo separate terminalN-acetylneuraminyl-α2,6-β-D-galactosyl-1,4-N-acetyl-β-D-glucosaminemoieties in one or more glycan portion(s) of the glycoprotein. In aspecific embodiment, only one α2,6 glycosidic bond in a is hydrolyzed.In a further specific embodiment, the bi-sialylated glycoprotein is abi-sialylated IgG immunoglobulin.

As an exemplary case, the IgG-Fc glycan G2 has two galactose moieties atthe termini of the antennate branches which can be sialylated. Undersuitable reaction conditions, the N-terminally truncated variant Δ89hST6Gal-I catalyzes the synthesis of IgG with bi-sialylated G2 glycans(G2+2SA) at the immunoglobulin Fc portion. However, upon accumulation of5′-CMP the enzyme variant acts as a sialidase catalyzing removal byhydrolysis of a sialic acid moiety from the bi-sialylated (G2+2SA)antibodies resulting in mono-sialylated (G2+1SA) antibodies. Thisproperty was found unexpectedly and appears to represent an intrinsicsialidase (neuraminidase) activity.

In a basic publication on human ST6Gal-I it was stated that the enzymedoes not contain any sialidase activity, see Sticher et al.Glycoconjugate Journal 8 (1991) 45-54. In view of the present surprisingfinding it becomes possible to preferentially synthesize glycoproteinswith mono-, bi- or even higher sialylated glycans, using the same enzymeand controlling the reaction kinetics of the enzyme by controlling CMPin the sialylation reaction mixture. A further advantage is that bothactivities, sialylation activity and sialidase activity are provided bythe same enzyme, in the same reaction vessel.

The general finding documented in the present disclosure is, however,that there exists a variant mammalian glycosyltransferase, specificallya glycosyltransferase according to the present disclosure, which iscapable of catalyzing hydrolysis of the α2,6 glycosidic bond of aN-acetylneuraminyl-α,2,6-β-D-galactosyl-1,4-N-acetyl-β-D-glucosaminemoiety of a glycan in a glycoprotein. In addition to the already knownsialyltransferase (sialylation) activity the surprising finding was thatat least as specifically shown for the N-terminally truncated humanp-galactoside-α-2,6-sialyltransferase I having the amino acid sequenceof SEQ ID NO:2 there is not only conventional sialyltransferase but alsosialidase enzymatic activity mediated by this enzyme. Interestingly, inthe exemplary cases these two activities were not observed at the sametime, which may partly explain the unexpected finding. Thus, in theabsence of CMP sialyltransferase activity dominates in the beginning andsialidase activity becomes apparent only at a later stage during theincubation, once sufficient amounts of CMP have accumulated.Nevertheless, the apparent recognition of two distinct activities of thesame enzyme allows to control the extent of sialylation of targetmolecules, e.g. by way of varying incubation time.

However, in a very elegant way, sialylation can be maximized by addingto the sialylation reaction mixture an enzyme capable of hydrolyzing thephosphoester bond in 5′-cytidine-monophosphate under the conditionswhich permit sialyltransferase enzymatic activity. Thus, the by-productCMP is removed and the sialylation catalysis by the sialyltransferase isnot counteracted.

Yet, another aspect and a specific embodiment of all other aspects asdisclosed herein is the use of an enzyme capable of hydrolyzing thephosphoester bond in 5′-cytidine-monophosphate under the conditionswhich permit sialyltransferase enzymatic activity to maintainglycosyltransferase activity and/or inhibit sialidase activity of avariant mammalian glycosyltransferase as disclosed herein, specificallythe N-terminally truncated human β-galactoside-α,-2,6-sialyltransferaseI having the amino acid sequence of SEQ ID NO:2.

Such controlled sialylation is provided as a novel means to synthesizein vitro mono-, bi-, and higher sialylated glycoproteins with a desireddegree of sialylation. Thus, though exemplified by showing the desiredtechnical effects with IgG molecules, the uses according to thedisclosures in here also allow to process other glycoproteins in asimilar way, with the proviso that concerning glysosyltransferaseactivity, the glycoproteins comprise two or more terminal antennateβ-D-galactosyl-1,4-N-acetyl-β-D-glucosamine moieties. The same reasoningapplies in an analogous way to glycolipids.

In a particular example, recombinant humanized IgG1 and IgG4 monoclonalantibodies (mabs), characterized as G2+0SA (=two acceptor sites present,no sialylation at any acceptor site), as well as EPO (=erythropoietin)were used as targets in sialylation experiments (30 μg enzyme/300 μgtarget protein). Δ89 hST6Gal-I was used under standard reactionconditions and the G2+0SA, G2+1SA (=mono-sialylation, one out of twoacceptor sites is sialylated) and G2+2SA (=bi-sialylation, both possibleacceptor sites are sialylated) status was analyzed by mass spectrometry.

Due to the high expression rates and the efficient purificationprocedures the exemplary Δ89 hST6Gal-I but also functionally equivalentenzymes can be made available in large quantities and with high purity.The variant Δ89 hST6Gal-I enzyme is active with high molecular weightsubstrates of which monoclonal antibodies are just one example.Depending on the incubation time, Δ89 hST6Gal-I in combination with aCMP-hydrolyzing enzyme shows good performance in sialylation experimentsusing monoclonal antibodies with bi-antennary glycan as substrate. Usingembodiments of the present disclosure the preferably bi-sialylatedglycans are obtained with great advantage after shorter incubationperiods, such as 8 hours. Tetra-antennary glycans are also accepted assubstrate (data not shown). The results demonstrate technical advantagefor in vitro glycoengineering of therapeutic antibodies.

The following items further provide specific aspects of the disclosure,and specific embodiments to practice the teachings provided herein.

-   1. Use of a soluble human β-galactoside-α,-2,6-sialyltransferase I    comprising the amino acid motif from position 90 to position 108 in    SEQ ID NO:1 for in vitro hydrolyzing in the presence of    5′-cytidine-monophosphate the α2,6 glycosidic bond in a    N-acetylneuraminyl-α,2,6-β-D-galactosyl-1,4-N-acetyl-β-D-glucosamine    moiety, the moiety being a terminal structure of a glycan in a    sialylated glycoprotein or glycolipid.-   2. The use according to item 1, wherein the soluble human    β-galactoside-α-2,6-sialyltransferase I comprises the amino acids    from position 90 to position 406 in SEQ ID NO:1.-   3. The use according to any of the items 1 and 2, wherein the amino    acid sequence of the soluble β-galactoside-α,-2,6-sialyltransferase    I is the amino acid sequence of SEQ ID NO:2.-   4. The use according to any of the items 1 to 3, wherein the    glycoprotein is selected from the group consisting of a cell surface    glycoprotein and a serum glycoprotein.-   5. The use according to item 4, wherein the serum glycoprotein is    selected from a glycosylated protein signaling molecule, a    glycosylated immunoglobulin, and a glycosylated protein of viral    origin.-   6. The use according to any of the items 1 to 5, wherein the    glycoprotein is recombinantly produced.-   7. The use according to item 6, wherein the glycoprotein is    recombinantly produced in a transformed host cell of mammalian    origin.-   8. The use according to any of the items 1 to 7, wherein the    glycoprotein is an immunoglobulin of human origin or a humanized    immunoglobulin, the immunoglobulin being selected from the group    consisting of IgG1, IgG2, IgG3, IgG4.-   9. The use according to any of the items 1 to 7, wherein the    glycoprotein is selected from EPO and asialofetuin.-   10. An aqueous composition comprising    -   (a) a soluble human β-galactoside-α-2,6-sialyltransferase I        comprising the amino acid motif from position 90 to position 108        in SEQ ID NO: 1;    -   (b) cytidine-5′-monophospho-N-acetylneuraminic acid, or a        functional equivalent thereof,    -   (c) a glycosylated target molecule selected from a glycoprotein        and a glycolipid, the target molecule comprising one or more        antenna(e), at least one antenna having as a terminal structure        a β-D-galactosyl-1,4-N-acetyl-β-D-glucosamine moiety with a        hydroxyl group at the C6 position in the galactosyl residue;    -   (d) an aqueous solution permitting glycosyltransferase enzymatic        activity;    -   wherein the aqueous composition further comprises an enzyme        capable of hydrolyzing the phosphoester bond in        5′-cytidine-monophosphate under conditions permitting        glycosyltransferase enzymatic activity.-   11. The aqueous composition according to item 10, wherein the    soluble human β-galactoside-α-2,6-sialyltransferase I comprises the    amino acids from position 90 to position 406 in SEQ ID NO:1.-   12. The aqueous composition according to any of the items 10 and 11,    wherein the amino acid sequence of the soluble    β-galactoside-α,-2,6-sialyltransferase I is the amino acid sequence    of SEQ ID NO:2.-   13. The aqueous composition according to any of the items 10 to 12,    wherein the glycosylated target molecule is a glycoprotein selected    from the group consisting of a cell surface glycoprotein and a serum    glycoprotein.-   14. The aqueous composition according to any of the items 10 to 13,    wherein the serum glycoprotein is selected from a glycosylated    protein signaling molecule, a glycosylated immunoglobulin, and a    glycosylated protein of viral origin.-   15. The aqueous composition according to any of the items 10 to 14,    wherein the glycoprotein is recombinantly produced.-   16. The aqueous composition according to item 15, wherein the    glycoprotein is recombinantly produced in a transformed host cell of    mammalian origin.-   17. The aqueous composition according to any of the items 10 to 16,    wherein the glycoprotein is an immunoglobulin of human origin or a    humanized immunoglobulin, the immunoglobulin being selected from the    group consisting of IgG1, IgG2, IgG3, IgG4.-   18. The aqueous composition according to any of the items 10 to 16,    wherein the glycoprotein is selected from EPO and asialofetuin.-   19. The aqueous composition according to any of the items 10 to 18,    wherein the aqueous solution comprises water, a buffer salt capable    of buffering in the pH range of pH 6 to pH 8, and optionally a    compound selected from the group consisting of a neutral salt, a    salt with a divalent cation, an antioxidant, a surfactant and a    mixture thereof.-   20. The aqueous composition according to any of the items 10 to 19,    the composition having a temperature of 0° C. to 40° C.-   21. The aqueous composition according to any of the items 10 to 20,    wherein the enzyme capable of the hydrolyzing phosphoester bond in    5′-cytidine-monophosphate is selected from the group consisting of    an alkaline phosphatase, an acid phosphatase, and a 5′ nucleotidase.-   22. The aqueous composition according to item 21, wherein the    alkaline phosphatase is selected from the group consisting of    alkaline phosphatase of bacterial origin, shrimp alkaline    phosphatase, calf intestine alkaline phosphatase, human placental    alkaline phosphatase, and a mixture thereof.-   23. The aqueous composition according to item 22, wherein the    aqueous composition further comprises Zn²⁺ ions.-   24. The aqueous composition according to item 21, wherein the 5′    nucleotidase is 5′nucleotidase CD73 of mammalian origin,    specifically of human origin.-   25. Use of an aqueous composition according to any of the items 10    to 24 for reducing 5′-cytidine-monophosphate-mediated inhibition and    thereby maintaining the sialylating activity of the soluble human    β-galactoside-α,-2,6-sialyltransferase I comprising the amino acid    motif from position 90 to position 108 in SEQ ID NO:1.-   26. The use according to item 25, wherein the sialylating activity    catalyzes transfer and covalent coupling of the sialic acid residue,    or the functional equivalent thereof, from the co-substrate to a    β-D-galactosyl-1,4-N-acetyl-β-D-glucosamine moiety with a hydroxyl    group at the C6 position in the galactosyl residue, the moiety being    a terminal structure of a glycan of a glycosylated target molecule    selected from a glycoprotein and a glycolipid.-   27. A method of producing in vitro a sialylated target molecule, the    method comprising the steps of    -   (a) providing an aqueous composition according to any of the        items 10 to 24;    -   (b) forming one or more terminal antennal        N-acetylneuraminyl-α2,6-β-D-galactosyl-1,4-N-acetyl-p-D-glucosamine        residue(s) by incubating the aqueous composition of step (a),        thereby reacting cytidine-5′-monophospho-N-acetylneuraminic        acid, or a functional equivalent thereof, as co-substrate,        thereby forming 5′-cytidine-monophosphate;    -   (c) hydrolyzing the phosphoester bond of the        5′-cytidine-monophosphate formed in step (b), thereby reducing        5′-cytidine-monophosphate-mediated inhibition, thereby        maintaining the activity of the soluble human        β-galactoside-α,-2,6-sialyltransferase I;    -   thereby producing in vitro the sialylated target molecule.-   28. The method according to item 27, wherein the method is performed    at a temperature of 0° C. to 40° C.-   29. The method according to any of the items 27 and 28, wherein    steps (b) and (c) are performed in the same vessel.-   30. The method according to any of the items 27 to 29, wherein    steps (b) and (c) are performed for a period selected from the group    consisting of 2 h to 96 h, 2 h to 23 h, 2 h to 6 h, and about 2 h.-   31. The method according to any of the items 27 to 29, wherein    steps (b) and (c) are performed for a period selected from the group    consisting of 6 h to 96 h, 6 h to 23 h, and about 6 h.-   32. The method according to any of the items 27 to 29, wherein    steps (b) and (c) are performed for a period selected from the group    consisting of 23 h to 96 h, and about 23 h.-   33. The method according to any of the items 27 to 29, wherein    steps (b) and (c) are performed for a period of about 96 h.-   34. Use of a soluble human β-galactoside-α,-2,6-sialyltransferase I    lacking the amino acid motif from position 90 to position 108 in SEQ    ID NO:1 for transferring in vitro and in the presence of    5′-cytidine-monophosphate a 5-N-acetylneuraminic acid residue from    the donor compound cytidine-5′-monophospho-N-acetylneuraminic acid,    or from a functional equivalent thereof, to an acceptor, the    acceptor being terminal β-D-galactosyl-1,4-N-acetyl-β-D-glucosamine    in a glycan moiety of a glycoprotein or a glycolipid.-   35. The use according to item 34, wherein the soluble human    β-galactoside-α-2,6-sialyltransferase I comprises the amino acids    from position 109 to position 406 in SEQ ID NO:1.-   36. The use according to any of the items 34 and 35, wherein the    amino acid sequence of the soluble    β-galactoside-α-2,6-sialyltransferase I is the amino acid sequence    of SEQ ID NO:5.-   37. The use according to any of the items 34 to 36, wherein the    glycoprotein is selected from the group consisting of a cell surface    glycoprotein and a serum glycoprotein.-   38. The use according to item 37, wherein the serum glycoprotein is    selected from a glycosylated protein signaling molecule, a    glycosylated immunoglobulin, and a glycosylated protein of viral    origin.-   39. The use according to any of the items 34 to 38, wherein the    glycoprotein is recombinantly produced.-   40. The use according to item 39, wherein the glycoprotein is    recombinantly produced in a transformed host cell of mammalian    origin.-   41. The use according to any of the items 34 to 40, wherein the    glycoprotein is an immunoglobulin of human origin or a humanized    immunoglobulin, the immunoglobulin being selected from the group    consisting of IgG1, IgG2, IgG3, IgG4.-   42. The use according to any of the items 34 to 40, wherein the    glycoprotein is selected from EPO and asialofetuin.-   43. An aqueous composition comprising    -   (a) a soluble human β-galactoside-α,-2,6-sialyltransferase I        lacking the amino acid motif from position 90 to position 108 in        SEQ ID NO:1;    -   (b) cytidine-5′-monophospho-N-acetylneuraminic acid, or a        functional equivalent thereof,    -   (c) a glycosylated target molecule selected from a glycoprotein        and a glycolipid, the target molecule comprising one or more        antenna(e), at least one antenna having as a terminal structure        a β-D-galactosyl-1,4-N-acetyl-β-D-glucosamine moiety with a        hydroxyl group at the C6 position in the galactosyl residue;    -   (d) an aqueous solution permitting glycosyltransferase enzymatic        activity;    -   wherein the aqueous composition further comprises        5′-cytidine-monophosphate.-   44. The aqueous composition according to item 43, wherein the    soluble human β-galactoside-α-2,6-sialyltransferase I comprises the    amino acids from position 109 to position 406 in SEQ ID NO:1.-   45. The aqueous composition according to any of the items 43 and 44,    wherein the amino acid sequence of the soluble    β-galactoside-α,-2,6-sialyltransferase I is the amino acid sequence    of SEQ ID NO:5.-   46. The aqueous composition according to any of the items 43 to 45,    wherein the glycosylated target molecule is a glycoprotein selected    from the group consisting of a cell surface glycoprotein and a serum    glycoprotein.-   47. The aqueous composition according to any of the items 43 to 46,    wherein the serum glycoprotein is selected from a glycosylated    protein signaling molecule, a glycosylated immunoglobulin, and a    glycosylated protein of viral origin.-   48. The aqueous composition according to any of the items 43 to 47,    wherein the glycoprotein is recombinantly produced.-   49. The aqueous composition according to item 48, wherein the    glycoprotein is recombinantly produced in a transformed host cell of    mammalian origin.-   50. The aqueous composition according to any of the items 43 to 49,    wherein the glycoprotein is an immunoglobulin of human origin or a    humanized immunoglobulin, the immunoglobulin being selected from the    group consisting of IgG1, IgG2, IgG3, IgG4.-   51. The aqueous composition according to any of the items 43 to 49,    wherein the glycoprotein is selected from EPO and asialofetuin.-   52. The aqueous composition according to any of the items 43 to 51,    wherein the aqueous solution comprises water, a buffer salt capable    of buffering in the pH range of pH 6 to pH 8, and optionally a    compound selected from the group consisting of a neutral salt, a    salt with a divalent cation, an antioxidant, a surfactant and a    mixture thereof.-   53. The aqueous composition according to any of the items 43 to 52,    the composition having a temperature of 0° C. to 40° C.-   54. A method of producing in vitro a sialylated target molecule, the    method comprising the steps of    -   (a) providing an aqueous composition according to any of the        items 43 to 53;    -   (b) forming one or more terminal antennal        N-acetylneuraminyl-α2,6-β-D-galactosyl-1,4-N-acetyl-β-D-glucosamine        residue(s) by incubating the aqueous composition of step (a),        thereby reacting cytidine-5′-monophospho-N-acetylneuraminic        acid, or a functional equivalent thereof, as co-substrate,        thereby forming 5′-cytidine-monophosphate;    -   (c) accumulating 5′-cytidine-monophosphate formed in step (b);    -   thereby producing in vitro the sialylated target molecule.-   55. The method according to item 54, wherein the method is performed    at a temperature of 0° C. to 40° C.-   56. The method according to any of the items 54 and 55, wherein    steps (b) and (c) are performed in the same vessel.-   57. The method according to any of the items 54 to 56, wherein    steps (b) and (c) are performed for a period selected from the group    consisting of 2 h to 72 h.-   58. Use of an enzyme capable of the hydrolyzing phosphoester bond in    5′-cytidine-monophosphate for maintaining sialyltransferase    enzymatic activity in a composition according to any of the items 10    to 24.-   59. Use of an enzyme capable of the hydrolyzing phosphoester bond in    5′-cytidine-monophosphate for inhibiting sialidase enzymatic    activity in a composition according to any of the items 10 to 24.-   60. A preparation of sialylated immunoglobulins, each immunoglobulin    having a plurality of acceptor sites for human    β-galactoside-α,-2,6-sialyltransferase I, wherein less than 25% of    the acceptor sites in the preparation of sialylated immunoglobulins    are not sialylated, and 75% or more are sialylated, wherein the    preparation is obtained by a method according to any of the items 27    to 33.-   61. The preparation according to item 60, wherein less than 20% of    the acceptor sites in the preparation of sialylated immunoglobulins    are not sialylated, and 80% or more are sialylated.-   62. The preparation according to item 60, wherein less than 10% of    the acceptor sites in the preparation of sialylated immunoglobulins    are not sialylated, and 90% or more are sialylated.

The Examples that follow are illustrative of specific embodiments of thedisclosure, and various uses thereof. They set forth for explanatorypurposes only, and are not to be taken as limiting the disclosure.

Example 1

Test for Sialyltransferase Enzymatic Activity

Asialofetuin (desialylated fetuin, Roche Applied Science) was used asacceptor and CMP-9-fluoro-NANA (CMP-9-fluoresceinyl-NeuAc) was used asdonor substrate (Brossmer, R. & Gross H. J. (1994) Meth. Enzymol. 247,177-193). Enzymatic activity of a sialyltransferase was determined bymeasuring the transfer of sialic acid from the donor compound toasialofetuin. The reaction mix (35 mM MES, pH 6.0, 0.035% TRITON™ X-100(C₁₆H₂₆O₂), 0.07% BSA) contained 2.5 μg of enzyme sample, 5 μLasialofetuin (20 mg/mL) and 2 μL CMP-9-fluoro-NANA (1.0 mg/mL) in atotal volume of 51 μL. The reaction mix was incubated at 37° C. for 30min. The reaction was stopped by the addition of 10 μL of the inhibitorCTP (10 mM). The reaction mix was loaded onto a PD10 desalting columnequilibrated with 0.1 M Tris/HCl, pH 8.5. Fetuin was eluted from thecolumn using the equilibration buffer. The fractions size was 1 mL. Theconcentration of formed fetuin was determined using a fluorescencespectrophotometer. Excitation wave length was 490 nm, emission wasmeasured at 520 nm. Enzymatic activity was expressed as RFU (relativefluorescence unit). 10,000 RFU/μg is equivalent to a specific activityof 0.0839 nmol/μg×min.

Example 2

SDS Gel Electrophoresis

Analytical SDS gel electrophoresis was carried out using NuPAGE gels(4-12%, Invitrogen). Samples (36 μL) were diluted with 12 μL NuPAGE LDSsample buffer (Invitrogen) and incubated for 2 min at 85° C. Aliquots,typically containing 5 μg protein were loaded on the gel. The gels werestained using SimplyBlue SafeStain (Invitrogen).

Example 3

N-Terminal Sequencing by Edman Degradation

The N-terminal sequences of expressed variants of human ST6Gal-I wereanalyzed by Edman degradation using reagents and devices obtained fromLife Technologies. Preparation of the samples was done as described inthe instruction manual of the Life Technologies ProSorb SamplePreparation cartridges (catalogue number 401950) and the LifeTechnologies ProBlott Mini PK/10 membranes (catalogue number 01194). Forsequencing the Procise Protein Sequencing Platform was used.

Example 4

Mass Spectrometry of Glycosylated Human ST6Gal-I Enzymes

The molecular masses of variants of human ST6Gal-I expressed in HEKcells were analyzed. Glycosylated forms of human ST6Gal-I were prepared,and prepared material was analyzed using Micromass Q-Tof Ultima andSynapt G2 HDMS devices (Waters UK) and MassLynx V 4.1 software.

For mass spectrometry measurement the samples were buffered inelectrospray medium (20% acetonitrile+1% formic acid). The bufferexchange was performed with Illustra™ MicroSpin™ G-25 columns(GE-Healthcare). 20 μg sialyltransferase variant with a concentration of1 mg/mL was applied to the pre-equilibrated column and eluated bycentrifugation. The resulting eluate was analyzed by electrosprayionization mass spectrometry.

Example 5

Mass Spectrometry of Deglycosylated Human ST6Gal-I Enzymes

The molecular masses of variants of human ST6Gal-I expressed in HEKcells were analyzed. Delycosylated forms of human ST6Gal-I were analyzedusing Micromass Q-Tof Ultima and Synapt G2 HDMS devices (Waters UK) andMassLynx V 4.1 software.

For deglycosylation samples of the sialyltransferase were denatured andreduced. To 100 μg sialyltransferase 45 μL denaturing buffer (6 Mguanidinium hydrochloride) and 13 μL TCEP(=tris(2-carboxyethyl)phosphine; 0.1 mM, diluted in denaturing buffer)were added. Further the appropriate volume of ultrapure water was added,so that the overall concentration of guanidinium hydrochloride was about4 M. After incubation of the sample for 1 h at 37° C. the buffer waschanged using a Bio-SpinR 6 Tris column (Bio Rad), which waspre-equilibrated with ultrapure water. The whole sample was applied ontothe column and eluted by centrifugation. To the resulting eluate 5.5 μLof 0.1 U/μL solution of PNGase-F was added and incubated at 37° C. overnight. Afterwards the samples were adjusted to 30% ACN (=acetonitrile)and 1% FA (formamide) and analyzed by electrospray ionization massspectrometry.

Example 6

Cloning of pM1MT Expression Constructs for Transient Expression inMammalian Host Cells of Truncated Variant Δ89 of Human ST6Gal-I

Truncated variant Δ89 of human ST6Gal-I was cloned for transientexpression using an Erythropoietin signal peptide sequence (Epo) and apeptide spacer of two amino acids (“AP”). For the Epo-AP-Δ89 hST6Gal-Iconstruct codon-optimized cDNAs were synthesized, see SEQ ID NO:3.Instead of the natural leader sequences and the N-terminal proteinsequences, the hST6Gal-I coding region harbors the Erythropoietin signalsequence plus AP linker sequence in order to ensure correct processingof expressed polypeptides by the secretion machinery of the host cellline. In addition, the expression cassettes features SaI and BamHIrestriction sites for cloning into the multiple cloning site of thepredigested pM1MT vector fragment (Roche Applied Science). Expression ofthe ST6Gal-I coding sequence is therefore under control of a humancytomegalovirus (CMV) immediate-early enhancer/promoter region, followedby an “intron A” for regulated expression, and a BGH polyadenylationsignal.

Expression of the Epo-AP-Δ89 hST6Gal-I construct in HEK cells, andsecretion of Δ89 hST6Gal-I protein into cell supernatant was performedas described in Example 8.

Example 7

Cloning of pM1MT Expression Constructs for Transient Expression inMammalian Host Cells of Truncated Variant Δ108 of Human ST6Gal-I

Truncated variant Δ108 of human ST6Gal-I was cloned for transientexpression using an Erythropoietin signal peptide sequence (Epo) and apeptide spacer of four amino acids (“APPR”). For the Epo-APPR-Δ108hST6Gal-I construct a codon-optimized cDNAs was synthesized, see SEQ IDNO:6. The natural hST6Gal-I-derived mRNA leader and N-terminal proteinsequences were exchanged with the Erythropoetin signal sequence and the“APPR” linker sequence to ensure correct processing of the polypeptideby the secretion machinery of the HEK host cell line. In addition, theexpression cassettes feature SalI and BamHI sites for cloning into themultiple cloning site of the pre-digested pM1MT vector fragment (RocheApplied Science). Expression of the hST6Gal-I coding sequence wasthereby put under the control of a human cytomegalovirus (CMV)immediate-early enhancer/promoter region; the expression vector furtherfeatured an “intron A” for regulated expression and a BGHpolyadenylation signal. Expression of the Epo-APPR-Δ108 hST6Gal-Iconstruct (SEQ ID NO:6) in HEK cells, and secretion of Δ108 hST6Gal-Iprotein into cell supernatant was performed as described in Example 8.

Example 8

Transformation HEK Cells and Transient Expression and Secretion

Transient gene expression (TGE) by transfection of plasmid DNA is arapid strategy to produce proteins in mammalian cell culture. Forhigh-level expression of recombinant human proteins a TGE platform basedon a suspension-adapted human embryonic kidney (HEK) 293 cell line wasused. Cells were cultured in shaker flasks at 37° C. under serum-freemedium conditions. The cells were transfected at approx. 2×10⁶ vc/mLwith the pM1MT expression plasmids (0.5 to 1 mg/L cell culture)complexed by the 293-Free™ (Merck) transfection reagent according to themanufacturer's guidelines. Three hours post-transfection, valproic acid,a HDAC inhibitor, was added (final conc. 4 mM) in order to boost theexpression (Backliwal et al. (2008), Nucleic Acids Research 36, e96).Each day, the culture was supplemented with 6% (v/v) of a soybeanpeptone hydrolysate-based feed. The culture supernatant was collected atday 7 post-transfection by centrifugation.

Example 9

Purification of the N-Terminal Truncation Variants of Human ST6Gal-Ifrom Supernatants of Transformed HEK Cells

HEK cells were transformed as described in Example 8. Expressionconstructs were prepared as described in Examples 6 and 7.

From supernatants of HEK cell fermentations the two enzyme variantsEpo-AP-Δ89 hST6Gal-I and Epo-APPR-Δ108 hST6Gal-I were purified using asimplified purification protocol. In a first step, a volume of 0.1 L ofculture supernatant was filtrated (0.2 μm), and the solution wasdialysed against buffer A (20 mM potassium phosphate, pH 6.5). Thedialysate was loaded onto a S-Sepharose™ ff (Fast Flow) column (1.6 cm×2cm) equilibrated with buffer A. After washing with 100 mL buffer A, theenzyme was eluted with a linear gradient of 10 mL buffer A and 10 mL ofbuffer A with 200 mM NaCl, followed by a wash step using 48 mL of bufferA with 200 mM NaCl. Fractions (4 mL) were analysed by an analytical SDSgel electrophoresis.

Fractions containing the Δ89 hST6Gal-I enzyme were pooled and dialyzedagainst buffer B (50 mM MES, pH 6.0). The dialyzed pool was loaded ontoa Heparin Sepharose ff column (0.5 cm×5 cm) equilibrated with buffer Band eluted using buffer B with 200 mM NaCl. Fractions (1 mL) containingthe enzyme were pooled and dialyzed against buffer B. Proteinconcentrations were determined at 280 nm (E280 nm [1 mg/mL]=1.931). Massspectrometry analysis showed that the recombinantly expressed Epo-AP-Δ89hST6Gal-I enzyme was secreted without the N-terminal amino acids AP.This finding was unexpected and indicated unusual cleavage of theexpressed protein by the signal peptidase while removing the Epoportion. For the recombinant human Δ89 hST6Gal-I enzyme a specificactivity of 3.75 nmol/pg×min was determined. FIG. 2 shows the results ofa SDS-PAGE of recombinant Δ89 hST6Gal-I variant purified from HEK cells.

Fractions containing the Δ108 hST6Gal-I enzyme were pooled and dialyzedagainst storage buffer (20 mM potassium phosphate, 100 mM sodiumchloride, pH 6.5). Protein concentration was determined at a wave lengthof 280 nm using a molar extinction coefficient of 1.871. Massspectrometric analysis of the recombinant protein secreted from the HEKcells transformed with the Epo-APPR-Δ108-hST6Gal-I expression constructconfirmed the N-terminal sequence “APPR”, thus indicating the expectedcleavage of the EPO signal sequence by the signal peptidase. For therecombinant human Δ108 hST6Gal-I variant from HEK cells a specificactivity of >600 RFU/pg was determined. FIG. 3 shows the results of aSDS-PAGE of recombinant Δ108 hST6Gal-I variant purified from HEK cells.

Example 10

Sialylation of Humanized Monoclonal Antibody IgG4 MAB Using Δ89hST6Gal-I

A highly galactosylated humanized monoclonal antibody IgG4 MAB was usedin sialylation experiments. The reaction mixture contained IgG4 MAB (300μg in 55 μL 35 mM sodium actetate/Tris buffer pH 7.0), the donorsubstrate CMP-NANA (150 μg in 50 μL water) and sialyltransferase (30 μgΔ89 hST6Gal-I in 20 mM potassium phosphate, 0.1 M NaCl, pH 6.5). Thesamples were incubated at 37° C. for a defined time. To stop thereaction the samples were frozen at −20° C. For mass analysis 100 μLdenaturing buffer (6 M guanidinium chloride) and 30 μL TCEP(=tris(2-carboxyethyl)phosphine; 0.1 mM, diluted in denaturing buffer)were added to the samples and the samples were incubated at 37° C. for 1h. The samples were buffered in electrospray medium [20% ACN(=acetonitrile), 1% FA (=formamide)] using pre-equilibrated Illustra™Nap5-Columns (GE-Healthcare). Samples were analyzed by electrosprayionization mass spectrometry and the content of G2+0SA, G2+1SA andG2+2SA N-glycans was determined. A Micromass Q-Tof Ultima and a SynaptG2 HDMS device (Waters UK) were used, the software used was MassLynx V4.1. To determine the kinetics of the sialylation the reaction wasincubated up to 72 h. FIG. 4 shows the relative amounts of differentlysialylated target proteins obtained after different time points duringthe incubation period.

The content of G2+0SA, G2+1SA and G2+2SA was determined by massspectrometry. For the variant Δ89 hST6Gal-I already after 2 h ofincubation a high content (88%) of the bi-sialylated form G2+2SA wasobtained, see FIG. 4 . The data also show that the content of G2+0SA andG2+1SA again increased over time due to the intrinsic CMP-dependentsialidase (neuraminidase) activity of Δ89 hST6Gal-I. After an incubationof 48 h a G2+1SA content of 71% was obtained.

FIG. 5 shows the spectra obtained by mass spectrometric analysis ofdifferent samples of IgG4 MAB. Samples were taken at time point t=0(lower panel), time point t=8 h (middle panel) and time point t=48 h(upper panel). The mass over charge (m/z) signals of one charge state inthe mass spectrum of the IgG molecule with G2+0SA, G2+1SA and G2+2SAglycans are depicted. The relative intensities of the differentsialylated species are derived from these signals. Corresponding to FIG.4 , at t=0 h G2+0SA is the major glycan species. At t=8 h the signal forG2+2SA is the dominant form whereas at t=48 h, G2+1SA is the mostabundant species. For the determined numerical values see FIG. 4 .

Example 11

Inhibition of Sialidase Activity of Δ89 hST6Gal-I by CTP

The compound cytidine-5′-triphosphate (CTP) is a known potent inhibitorof sialyltransferases (Scudder P R & Chantler E N BBA 660 (1981)136-141). To demonstrate that the sialidase activity is an intrinsicactivity of Δ89 hST6Gal-I, inhibition experiments were performed. In thefirst phase of the experiment the sialylation of IgG4 MAB by Δ89hST6Gal-I was performed to achieve a high content of G2+2SA (see Example10). After 7 h of incubation the G2+2SA content was 94%. Subsequently,CTP was added to inhibit the sialidase activity of Δ89 hST6Gal-I (finalconcentration of CTP: 0.67 mM). At different times samples were takenand the content of G2+0SA, G2+1SA and G2+2SA was determined by massspectrometry. The results are shown in FIG. 6 . Compared toinhibitor-free conditions shown in FIG. 4 the degradation of G2+2SAcaused by the sialidase activity was significantly reduced. After 72 hof incubation 73% of G2+2SA were still present. The inhibition of thesialidase activity by a known inhibitor of sialyltransferase activitystrongly indicates that both activities are located in the same activecenter of Δ89 hST6Gal-I.

Example 12

Sialylation of IgG1 MAB with Δ89 hST6Gal-I

The amount of 15 mg of a highly galactosylated humanized monoclonalantibody IgG1 MAB was used for sialylation treatment. The reactionmixture contained defined amounts of IgG1 MAB (15 mg in 1,854 μL aqueousbuffer containing 20 mM sodium actetate, 50 mM Tris buffer, pH 8.0), thedonor substrate CMP-NANA (7,500 μg in 2,500 μL water) andsialyltransferase (1,500 μg recombinantly produced and purified Δ89hST6Gal-I in 202 μL of 20 mM potassium phosphate, 0.1 M NaCl, pH 6.5).The components were mixed and the resulting reaction mixture wasincubated at 37° C. for different times, including 2 h, 4 h, 8 h, 24 h,and 48 h. Purification of sialylated antibody was performed as inExample 13.

To analyze the degree of sialylation, 124 μL denaturing buffer (6 MGuanidinium chloride in water) and 30 μL TCEP(=tris(2-carboxyethyl)phosphine; 0.1 mM, diluted in denaturing buffer)were added to 76 μL (corresponding to 250 μg IgG1 MAB) and the samplewas incubated at 37° C. for 1 h. After that the sample was buffered inelectrospray medium [20% ACN (=acetonitrile), 1% FA (=formamide)] usingpre-equilibrated ILLUSTRA™ NAP™5-Columns (GE-Healthcare). Subsequentlythe sample was analyzed by electrospray ionization mass spectrometry,and the content of G2+0SA, G2+1SA and G2+2SA N-glycans was determined. ASynapt G2 HDMS device (Waters UK) was used, the software used wasMassLynx V 4.1.

Example 13

Purification of Sialylated IgG1 MAB

To remove sialyltransferase and CMP-NANA from the incubated sialylationreaction mixture of Example 12, incubated IgG1 MAB was purified usingProtein A. The reaction mixture was applied to a Protein A columnequilibrated with 25 mM Tris, 25 mM NaCl, 5 mM EDTA(=ethylenediaminetetraacetic acid), pH 7. The column was washed with 25mM Tris, 25 mM NaCl, 500 mM TMAC (=tetramethylammonium chloride), 5 mMEDTA pH 5.0 and then with 25 mM Tris, 25 mM NaCl, 5 mM EDTA pH 7.1. IgG1MAB was eluted with 25 mM Na-Citrate. To avoid spontaneous desialylationat low pH, the pH was adjusted to pH 7.0 using 1 M Tris pH 9.0. Usingthis procedure sialylated IgG1 MAB was obtained in pure form, with atypical yield of 12 mg.

Example 14

Sialidase Activity of Δ89 hST6Gal-I on IgG1 MAB in the Presence andAbsence of CMP

Cytidine monophosphate (5′-CMP, =CMP) is a product of the reactioncatalyzed by sialyltransferase enzymes, generated in the course of theglycosyltransferase reaction from the co-substrate CMP-NANA. Withincubation time of a sialylation reaction CMP accumulates in thereaction mixture. To demonstrate that the inherent sialidase activity isCMP-dependent, highly sialylated IgG1 monoclonal antibody IgG1 MABG2+2SA was prepared by incubation with Δ89 hST6Gal-I in the presence ofCMP-NANA, as described in Example 12, and purified as described inExample 13.

To an amount of 1,250 μg (in 194 μL) highly sialylated IgG1 MABaccording to Example 12 with an incubation period for sialylation of 8h, 125 μg sialyltransferase variant (30 μg/300 μg IgG1 MAB) was added.

Different N-terminally truncated hST6Gal-I enzyme variants were testedfor CMP-dependent sialidase activity:

-   -   Δ89 hST6Gal-I (Example 9)    -   Δ108 hST6Gal-I (Example 9)    -   Δ57 hST3Gal-I (obtained from R&D Systems)

Four different experiments were made using Δ89 hST6Gal-I (16.8 μL with125 μg), Δ108 hST6Gal-I (17.3 μL with 125 μg), Δ57 hST3Gal-I (20.1 μLwith 125 μg) and a negative control (no enzyme, 20.1 μL ultrapurewater). The enzymes were tested for sialidase activity in the absenceand presence of CMP (10-fold excess based on molarity). Theconcentrations were as shown as follows:

Δ89 hST6Gal-I (16.8 μL with 125 μg): 11.8 μg CMP (c=0.5 mg/mL 23.6 μL)

Δ108 hST6Gal-I (17.3 μL with 125 μg): 12.3 μg CMP (c=0.5 mg/mL 24.5 μL)

Δ57 hST3Gal-I (20.1 μL with 125 μg): 12.3 μg CMP (c=0.5 mg/mL 24.5 μL)

Negative control (no enzyme): 12.3 μg CMP (c=0.5 mg/mL 24.5 μL)

The samples were incubated at 37° C. in 20 mM sodium citrate, 35 mM TrispH 6.5. Aliquots were taken as samples after different incubation times,and were analyzed.

To analyze the degree of sialylation of IgG1 MAB in the samples, 124 μLdenaturing buffer (6 M Guanidinium chloride in water) and 30 μL TCEP(=tris(2-carboxyethyl)phosphine; 0.1 mM, diluted in denaturing buffer)were added to 76 μL (corresponding to 250 μg IgG1 MAB) and the samplewas incubated at 37° C. for 1 h. After that the sample was buffered inelectrospray medium (20% ACN (=acetonitrile), 1% FA (=formamide)) usingpre-equilibrated ILLUSTRA™ NAP™5-Columns (GE-Healthcare). Subsequentlythe sample was analyzed by electrospray ionization mass spectrometry andthe content of G2+0SA, G2+1SA and G2+2SA N-glycans was determined. ASynapt G2 HDMS device (Waters UK) was used, the software used wasMassLynx V 4.1.

The results are shown in FIGS. 7-10 . In the reaction mixture withoutCMP no degradation of G2+2SA was observed even after incubation for 46 h(FIG. 7 ). Whereas in the presence of CMP a degradation of G2+2SA wasmeasured accompanied by an increase of the content of G2+1SA (FIG. 8 ).Under the conditions described above Δ89 hST6Gal-I showed aCMP-dependent sialidase activity, whereas Δ108 hST6Gal-I (FIG. 9 ) andΔ57 ST3Gal-I (FIG. 10 ) did not show any sialidase activity in thepresence of CMP. In the latter case this is noted that the enzyme isspecific for 2-3 glycosidic bonds.

Example 15

Sialylation of IgG4 MAB Using Δ89 hST6Gal-I in the Presence ofPhosphatase Enzymatic Activity

Suppression of CMP-dependent sialidase activity of Δ89 hST6Gal-I wasstudied by continuous removal of CMP formed during the reaction.

In the experiments the enzymes (i) 5′-nucleotidase (EC 3.1.3.5) having awide specificity for 5′-nucleotides, and (ii) alkaline phosphatase (EC3.1.3.1) (both provided by commercial suppliers) were used. Theparticular 5′-nucleotidase used here is also known asecto-5′-nucleotidase or CD73 (Cluster of Differentiation 73), in humansencoded by the NT5E gene. Both enzymes dephosphorylate CMP, i.e.catalyze hydrolysis of the phosphoester bond in CMP. In the experimentsof this Example the enzymes were used to degrade CMP generated by thesialyltransferase reaction from the co-substrate CMP-NANA. In theabsence of CMP no intrinsic sialidase activity of Δ89 hST6Gal-I wasobserved.

To 1,000 μg IgG4 MAB (182 μL) 500 μg CMP-NANA (3 mg/mL, 166.7 μL), 100μg Δ89 hST6Gal-I (13.4 μL, 30 μg/300 μg IgG4 MAB) and different amountsof nucleotidase (Nu) and alkaline phosphatase (AP) were added. As Zn²⁺ions are essential for the activity of AP, these were added to a finalconcentration of 0.1 mM). The buffer used was 20 mM sodium acetate/Tris,pH 6.5.

Different amounts of the enzymes were added to the reaction mixtures tostudy the effect of the dephosphorylating enzymes:

-   -   1) 5′-nucleotidase CD73 was used in a concentration of 0.1        μg/μL. To the reactions 0.10 μg, 0.25 μg and 0.50 μg were added.    -   2) Alkaline phosphatase (AP) was used in a concentration of 1        μg/μL and 10 μg/μL. To the reactions 1 μg, 5 μg, 10 μg and 100        μg were added.

After addition of the respective amounts of enzymes the samples wereincubated at 37° C. Samples were taken at several time points to controlthe degree of sialylation. Therefore 110 μL denaturing buffer (6 MGuanidinium chloride) and 30 μL TCEP (0.1 mM, diluted in denaturingbuffer) were added to 90 μL of the sample (about 250 μg IgG4 MAB) andthe sample was incubated at 37° C. for 1 h. After that the sample wasbuffered in electrospray medium [20% ACN (=acetonitrile), 1% FA(=formamide)] using pre-equilibrated ILLUSTRA™ NAP™5-Columns(GE-Healthcare). Then the sample was analyzed by electrospray ionizationmass spectrometry, and the content of G2+0SA, G2+1SA and G2+2SAN-glycans was determined. A Synapt G2 HDMS device (Waters UK) were used,the software used was MassLynx V 4.1.

Results for sialylation of IgG4 MAB by Δ89 hST6Gal-I in the absence orpresence of 5′-nucleotidase CD73 are depicted in FIGS. 11-14 ; andresults for sialylation of IgG4 MAB by Δ89 hST6Gal-I in the absence orpresence of alkaline phosphatase are depicted in FIGS. 15-19 . As itturned out, introducing a phosphatase activity capable of hydrolyzingthe phosphoester bond in 5′-CMP effectively reduced CMP-mediatedsialidase activity and promoted sialyltransferase activity.

The invention claimed is:
 1. A method of producing in vitro a sialylatedtarget molecule comprising: (a) providing an aqueous compositioncomprising (i) a soluble human β3-galatoside-α-2,6-sialyltransferase Icomprising an amino acid motif from position 90 to position 108 in SEQID NO:1; (ii) cytidine-5′-monophospho-N-acetylneuraminic acid; (iii) aglycosylated target molecule selected from the group consisting of aglycoprotein and a glycolipid, the target molecule comprising one ormore antenna(e), at least one antenna having as a terminal structure aβ-D-galactosyl-1,4-N-acetyl-β-D-glucosamine moiety with a hydroxyl groupat the C6 position in the galactosyl residue; (iv) an aqueous solutionpermitting glycosyltransferase enzymatic activity comprising a buffersalt; wherein the aqueous composition further comprises an enzymeselected from the group consisting of an alkaline phosphatase, an acidphosphatase and a 5′ nucleotidase that hydrolyzes the phosphoester bonein 5′-cytidine-monophosphate under conditions permittingglycosyltransferase enzymatic activity; (b) forming one or more terminalantennalN-acetylneuraminyl-α2,6-β-D-galactosyl-1,4-N-acetyl-β-D-glucosamineresidue(s) by incubating the aqueous composition of step (a), therebyreacting cytidine-5′-monophospho-N-acetylneuraminic acid asco-substrate, thereby forming 5′-cytidine-monophosphate; and (c)hydrolyzing the phosphoester bond of the 5′-cytidine-monophosphateformed in step (b), thereby reducing 5′-cytidine-monophosphate-mediatedinhibition, thereby maintaining the activity of the soluble humanβ-galactoside-α-2,6-sialyltransferase I; thereby producing in vitro thesialylated target molecule.
 2. The method of claim 1, wherein the methodis performed at a temperature of 0° C. to 40° C.
 3. The method of claim1, wherein steps (b) and (c) are performed in the same vessel.
 4. Themethod of claim 1, wherein steps (b) and (c) are performed for a periodselected from the group consisting of 2 h to 96 h, 2 h to 23 h, 2 h to 6h, and about 2 h.
 5. The method of claim 1, wherein steps (b) and (c)are performed for a period selected from the group consisting of 6 h to96 h, 6 h to 23 h, and about 6 h.
 6. The method of claim 1, whereinsteps (b) and (c) are performed for a period selected from the groupconsisting of 23 h to 96 h, and about 23 h.
 7. The method of claim 1,wherein steps (b) and (c) are performed for a period of about 96 h.